Blood vessels regulate primary motor neuronal pathfinding in zebrafish via exosome contained microRNA-22

A precise neuro-vascular communication is crucial to orchestrate directional migration and patterning of the complex vascular network and neural system. However, how blood vessels are involved in shaping the proper neuronal formation has not been fully understood. So far, limited studies have reported the discovery and functions of microRNAs (miRNAs) in guiding vascular and neural pathfinding. Currently, we showed that the deficiency of miRNA-22a, an endothelial-enriched miRNA, caused dramatic pathfinding defects both in intersegmental vessels (ISVs) and primary motor neurons (PMNs) in zebrafish embryos. Furthermore, we found the specific inhibition of miR-22a in ECs resulted in the patterning defects of both ISVs and PMNs. However, neuronal block of miR-22a mainly led to the axonal defects of PMN. Then we demonstrated that endothelial miR-22a regulates PMNs axonal navigation via exosome pathway. Sema4c was identified as a potential target of miR-22a through transcriptomic analysis and in silico analysis. Furthermore, luciferase assay and EGFP sensor assay in vivo confirmed the binding of miR-22a with 3’-UTR of sema4c. In addition, Down-regulation of sema4c in the miR-22a morphants significantly neutralized the aberrant patterning of vascular and neural networks. Our study revealed that miR-22a acted as a dual guidance cue coordinating vascular and neuronal patterning and expanded the repertoire of guidance molecules, which might be of use therapeutically to guide vessels and nerves in the relevant diseases that affect both systems.

Harmonization of L1CAM expression facilitates axon outgrowth and guidance of a motor neuron

ABSTRACTBrain development requires precise regulation of axon outgrowth, guidance and termination by multiple signaling and adhesion molecules. How the expression of these neurodevelopmental regulators is transcriptionally controlled is poorly understood. The Caenorhabditis elegans SMD motor neurons terminate axon outgrowth upon sexual maturity and partially retract their axons during early adulthood. Here we show that C-terminal binding protein 1 (CTBP-1), a transcriptional corepressor, is required for correct SMD axonal development. Loss of CTBP-1 causes multiple defects in SMD axon development: premature outgrowth, defective guidance, delayed termination and absence of retraction. CTBP-1 controls SMD axon guidance by repressing the expression of SAX-7, an L1 cell adhesion molecule (L1CAM). CTBP-1-regulated repression is crucial because deregulated SAX-7/L1CAM causes severely aberrant SMD axons. We found that axonal defects caused by deregulated SAX-7/L1CAM are dependent on a distinct L1CAM, called LAD-2, which itself plays a parallel role in SMD axon guidance. Our results reveal that harmonization of L1CAM expression controls the development and maturation of a single neuron.

An autism-associated calcium channel variant causes defects in neuronal polarity and axon termination in the ALM neuron of C. elegans

Variants of the CACNA1C voltage-gated calcium channel gene have been associated with autism and other neurodevelopmental disorders including bipolar disorder, schizophrenia, and ADHD. The Timothy syndrome mutation is a rare de novo gain-of-function variant in CACNA1C that causes autism with high penetrance, providing a powerful avenue into investigating the role of CACNA1C variants in neurodevelopmental disorders. In our previous work, we demonstrated that an egl-19(gof) mutation, that is equivalent to the Timothy syndrome mutation in the human homolog CACNA1C, can disrupt termination of the PLM axon in C. elegans. Here, we find that the egl-19(gof) mutation disrupts the polarity of process outgrowth in the ALM neuron of C. elegans. We also find that the egl-19(gof) mutation can disrupt termination of the ALM axon. These results suggest that the Timothy syndrome mutation can disrupt multiple steps of axon development. Further work exploring the molecular mechanisms that underlie these perturbations in neuronal polarity and axon termination will give us better understanding to how variants in CACNA1C contribute to the axonal defects that underlie autism.

Harmonization of L1CAM Expression Facilitates Axon Outgrowth and Guidance of a Motor Neuron

ABSTRACTBrain development requires precise regulation of axon outgrowth, guidance and termination by multiple signaling and adhesion molecules. How the expression of these neurodevelopmental regulators is transcriptionally controlled is poorly understood. The Caenorhabditis elegans SMD motor neurons terminate axon outgrowth upon sexual maturity and partially retract their axons during early adulthood. Here we show that C-Terminal Binding Protein-1 (CTBP-1), a transcriptional corepressor, is required for correct SMD axonal development. Loss of CTBP-1 causes multiple defects in SMD axon development: premature outgrowth, defective guidance, delayed termination and absence of retraction. CTBP-1 controls SMD axon development by repressing the expression of SAX-7 – a L1 cell adhesion molecule (L1CAM). CTBP-1-regulated repression is crucial as deregulated SAX-7/L1CAM causes aberrant SMD axons. We found that axonal defects caused by SAX-7/L1CAM misexpression are dependent on a distinct L1CAM, called LAD-2, which itself plays a parallel role in SMD axon guidance. Our results reveal that harmonization of L1CAM expression controls the development and maturation of a single neuron.

Retrograde Ret signaling controls sensory pioneer axon outgrowth

The trafficking mechanisms and transcriptional targets downstream of long-range neurotrophic factor ligand/receptor signaling that promote axon growth are incompletely understood. Zebrafish carrying a null mutation in a neurotrophic factor receptor, Ret, displayed defects in peripheral sensory axon growth cone morphology and dynamics. Ret receptor was highly enriched in sensory pioneer neurons and Ret51 isoform was required for pioneer axon outgrowth. Loss-of-function of a cargo adaptor, Jip3, partially phenocopied Ret axonal defects, led to accumulation of activated Ret in pioneer growth cones, and reduced retrograde Ret51 transport. Jip3 and Ret51 were also retrogradely co-transported, ultimately suggesting Jip3 is a retrograde adapter of active Ret51. Finally, loss of Ret reduced transcription and growth cone localization of Myosin-X, an initiator of filopodial formation. These results show a specific role for Ret51 in pioneer axon growth, and suggest a critical role for long-range retrograde Ret signaling in regulating growth cone dynamics through downstream transcriptional changes.

Intron-targeted mutagenesis reveals roles for Dscam1 RNA pairing-mediated splicing bias in neuronal wiring

Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam1) can potentially generate 38,016 different isoforms through stochastic, yet highly biased, alternative splicing. Genetic studies demonstrated that stochastic expression of multiple Dscam1 isoforms provides each neuron with a unique identity for self/non-self-discrimination. However, due to technical obstacles, the functional significance of the highly specific bias in isoform expression remains entirely unknown. Here, we provide conclusive evidence that Dscam1 splicing bias is required for precise mushroom body (MB) axonal wiring in flies in a variable exon-specific manner. We showed that targeted deletion of the intronic docking site perturbed base pairing-mediated regulation of inclusion of variable exons. Unexpectedly, we generated mutant flies with normal overall Dscam1 protein levels and an identical number but global changes in exon 4 and exon 9 isoform bias (DscamΔ4D−/− and DscamΔ9D−/−), respectively. DscamΔ9D−/− mutant exhibited remarkable mushroom body defects, which were correlated with the extent of the disrupted isoform bias. By contrast, the DscamΔ4D−/− animals exhibited a much less severe defective phenotype than DscamΔ9D−/− animals, suggestive of a variable domain-specific requirement for isoform bias. Importantly, mosaic analysis revealed that changes in isoform bias caused axonal defects but did not influence the self-avoidance of axonal branches. We concluded that, in contrast to the Dscam1 isoform number that provides the molecular basis for neurite self-avoidance, isoform bias may play a non-repulsive role in mushroom body axonal wiring.